Medical material and prosthetic skin in which cells can invade

ABSTRACT

Disclosed is a cell-penetrable medical material in which cells can invade, comprising denatured collagen with helix content of 0 to 80%, and carrier substance having higher resistance to enzyme decomposition than the denatured collagen. An artificial skin in which this medical material is used as the wound contact layer, and its preferred manufacturing methods are also disclosed.

TECHNICAL FIELD

The present invention relates to cell-penetrable medical material easilyassimilable into living tissues and useful as artificial tissues.Further, the present invention also relates to application of thismedical material to artificial skin.

BACKGROUND ART

Implantation is very effective for treating deficiency occurred in partof tissues or irreversible function deficiency. For avoiding the problemof immune incompatibility known as rejection, in this case, it ispreferable to transplant the tissue coming from other regions of thepatient or his relatives, namely allotransplantation. However, suchfavorable implanting tissues are not always available. So, such studiesfor providing implantable artificial tissues have heretofore been made.

First approach for getting artificial tissues free of rejection is toprovide the material having low histological reactivity, namely thematerial which fails to sensitize the tissue and immunocellular system.An example of this approach is the study to enhance hydrophobicity ofthe synthetic polymer material represented by polyurethanes.

Second approach is to provide the material which is capable ofassimilating rapidly into the tissue before inducing the immunoreactionthereby functioning as a part of an organ. More particularly, it is toconstruct the tissue similar to connective tissues by penetrating such acell having the tissue-healing function as fibroblast into the materialcoming from living bodies such as collagen. Since the new tissue thusformed is no longer not-self, no immune incompatibility would takeplace. Therefore, it can be said that this approach is more ideal thanthe first approach.

However, said second approach has the following defects.

Artificial materials consisting of collagen or the like derived fromliving bodies show high affinity to cellular tissues, but would beeasily decomposed by collagenase or other enzymes within the livingbodies. Therefore, there cannot be sufficiently kept the time for thepenetration of fibroblast or the like to construct new tissues. So it isnecessary to enforce the physical properties of the material byintroducing cross-linking with any means, in order that the material mayresist against the decomposition due to collagenase. Dehydratingcross-linking under heating or chemical cross-linking with chemicals canbe adopted therefore. Of these cross-linking methods, the dehydratingcross-linking is safer than the chemical treatment, but less resistantagainst collagenase than the chemical cross-linking. Therefore, it isgeneral that the chemical cross-linking is adopted singly, otherwise acombination of the chemical cross-linking and the dehydratingcross-linking is adopted.

Resistance against collagenase is markedly improved by introducing thecross-linking structure by said methods. For example, when thecross-linking structure is introduced into a collagen by merelydehydrating the collagen under heating at 110° C. in vacuum for 24hours, the cross-linked collagen is dissolved within one day by allowingto stand at 37° C. in 3 unit/ml collagenase solution. On the contrary,the collagen obtained by introducing the cross-linking structure with anisocyanate type cross-linking agent does not show any change onappearance in 7 days even by allowing to stand still at 37° C. in 1000unit/ml collagenase solution.

On the other hand, introduction of said strong cross-linking structurewould lower markedly the good affinity to cells or tissues, which is aninherent property of the collagen. Therefore, the penetration of cellswould be inhibited to accompany a problem that the new desired tissuescould not be formed.

As stated above, it is difficult to suffice both the requirement of goodresistance to enzymes and the requirement of good affinity to cells ortissues in materials derived from living bodies such as collagen.Therefore, while the second approach is very attractive, no medicalmaterial sufficing the requirements has been developed yet.

The cell-penetrable medical material may be considered to be very usefulas coating material for imbedding artificial organs or artificialvessels. However, its use as artificial skin is more realizable andeffective.

Artificial skin is an artificial medical material used for coatingtemporarily or eternally the injured region in order to preventbacterial infections or overflowing of the humor when any dermal tissueis injured by burning or ambustion. Thus, artificial skin a replacementof autograft skin.

As a wound coating material which can be used for the same purpose as inartificial skin, gauze, absorbent cotton and the like are heretoforused. However, these materials have a disadvantage that they have lowinhibitory ability against bacterial infections. In addition, becausethey absorb rapidly the exudate, the surface of wound would be dried somuch and accompanied by pain, bleeding or the like in peeling them.Although sometimes an ointment or the like is used together for avoidingsaid problem, in this case there would take place another disadvantagethat the exudate is so insufficiently absorbed that the surface of woundbecome excessively moist.

When the surface of wound extends in a broad scope, the followingcoating films are used. First category includes silicone gauze, siliconerubber film, synthetic fiber sheet such as nylon, teflon or the likehaving velour-like superficial structure, and other synthetic materials.Second category includes lyophilizated pig's skin, chitin unwoven cloth,collagen film, polyamino acid sponge, mucopolysaccharide complexcollagen film, and other materials derived from living bodies.

However, coating films made from said synthetic materials havedisadvantages such as poor tight adhesion to the injured region and lowsteam permeability together with easy inclination to induce cracking.Further, said coating films derived from living bodies showcomparatively better adaptability to living bodies but have difficultyin availability of raw material. Moreover, most of them haveantigenicity as well as defects such as inclination to deteriorate bybacterial infection or contact with the exudate.

In addition to said coating films, certain complex film made fromcollagen-treated nylon mesh and silicone film has recently beendeveloped and is commercially available. This complex film has favorableclose adhesion to the surface of wound and appropriate waterpermeability. However, the complex film adheres to the surface of wound,because the granulation tissue enters into the nylon mesh in the courseof curing. Since the nylon mesh remains in the granulation tissuewithout decomposition, after curing the complex film must be peeledtogether with remarkable pain.

DISCLOSURE OF THE INVENTION

A first object of the present invention is to provide cell-penetrablemedical material which, because of having the desired resistance todecomposing enzymes, can keep the necessary mechanical strength over acertain period under in vivo circumstances together with favorableaffinity to cells and tissues, and also to provide a process forpreparing said material.

A second object of the present invention is to provide artificial skinwhich has a satisfactory function as a replacement of autograft skin,using said medical material, and also to provide a process for preparingit. Thus, artificial skin of the present invention can inhibit bacterialinfection and overflowing of the body fluid by coating temporarily oreternally the surface of wound and ca accelerate sufficiently repairingthe tissue due to cell growth.

The cell-penetrable medical material of the present invention comprisesdenatured collagen with helix content of 0 to 80%, and carrier substancehaving higher resistance against enzyme decomposition than the denaturedcollagen.

The first artificial skin of the present invention comprises a supportlayer composed of fibroin, a wound contact layer laminated on one sideof the support layer, and moisture permeation regulating layer forcontrolling moisture permeation which is laminated on the other side ofthe support layer, wherein the wound contact layer contains denaturedcollagen with helix content of 0 to 80% and fibrous collagen. Thefibrous collagen has a higher resistance against enzyme decompositionthan the denatured collagen. That is, the fibrous collagen functions asthe carrier substance of the denatured collagen. It is preferable toincorporate antibacterial agent into at least one layer of wound contactlayer, support layer and moisture permeation regulating layer.

The second artificial skin of the present invention comprises a supportlayer composed of fibrous collagen matrix possessing crosslinkingstructure, a wound contact layer laminated on one side of the supportlayer, and a moisture permeation regulating layer for controlling themoisture penetration which is laminated on the other side of the supportlayer, wherein the wound contact layer contains denatured collagen withhelix content of 0 to 80% and fibrous collagen. It is preferable toincorporate antibacterial agent into at least one layer of wound contactlayer, support layer and moisture permeation regulating layer.

The method of manufacturing artificial skin of the present invention isfor obtaining the foregoing first or second artificial skin. This methodcomprises the steps of:

a step of preparing a mixture solution containing denatured collagenwith helix content of 0 to 80%, and fibrous collagen,

a step of placing a fibroin membrane or a fibrous collagen membranehaving crosslinking structure on the liquid surface of the mixturesolution, and then performing lyophilization, thereby forming a laminateof a support layer composed of the fibroin membrane or the fibrillatedcollagen membrane, and a wound contact layer made of porous substancecontaining the denatured collagen and the fibrous collagen,

a step of forming a viscous thin film made of a substance for providinga moisture permeation membrane, on a substrate having a peeling surface,

a step of placing the surface of the support layer on the viscous thinmembrane, and

a step of drying the viscous thin film until hardened, and then heatingfor 1 to 24 hours at 50° to 180° C. in a vacuum of less than 0.05 Torr.

It is preferable to incorporate an antibacterial agent into at least oneof the solution containing denatured collagen and fibrous collagen, thefibroin membrane or the fibrous collagen membrane having crosslinkingstructure, and the viscous thin film for providing the moisturepermeation regulating layer.

The invention is described in further detail below.

CELL-PENETRABLE MEDICAL MATERIAL

The cell-penetrable medical material of the present invention isrealized on the basis of the discovery that cells can easily invade intoa collagen layer in which collagen has been denatured up to the helixcontent of 0 to 80%.

To begin with, the denatured collagen with helix content of 0 to 80% isexplained below. The helix content refers to the content of the triplechain helix which is characteristic of collagen. In the denaturedcollagen, any part or all of the triple chain helix is transformed intorandom coil. Therefore, [1-helix content] corresponds to the degree ofdenaturing. The denatured collagen is obtained by heating, chemicaltreatment, or physical treatment of collagen. The most preferable methodfor obtaining the denatured collagen is heat treatment.

Such denatured collagen is prepared, for example, in the followingmanner. The collagen material derived from the bovine corium is treatedin acid or alkali, and collagen composed of triple chain helix isobtained. Successively, in the presence of water, the collagen thusobtained is heated for 20 minutes to 24 hour at 50 to 125° C.,preferably 90° to 121° C., thereby denaturing the triple chain helixinto random coil. For example, when heated for 30 minutes at 60° C., thehelix content becomes about 40%. Or when heated for 24 hours at 100° C.,the helix content will be 0%. The helix content can be measured by thecircular dichromism spectrometer (CD) or infrared spectrophotometer (P.L. Gorden et al., Macromoles, 1(6), 954, 1974; M. Nakura, Hashimoto, etal., High Polymer Article Collection, 41(8), 473, 1984). The values ofhelix content cited in this specification are the results calculated onthe basis of spectroscopic measurements. Incidentally, from the resultsof test on electrophoresis, it has been found that part of collagenmolecule was cleaved in this denatured collagen.

The denatured collagen used in this invention has the helix content of 0to 80% as mentioned above, or preferably 0 to 50%. It is also desired touse the denatured collagen in which the antigenic portion (teropeptide)at the terminal of the collagen molecule has been removed. Such collagenlack of antigenicity and free of this teropeptide is generally known bythe tradename of Aterocollagen. Aterocollagen is obtained by treatingthe starting collagen with acid or alkali, and then treating with pepsinwhich specifically acts on the teropeptide.

Since the helix of the denatured collagen used in the medical materialof the invention is transformed into random coil and partly cleaved onthe way, it seems that cells can invade easily into the denaturedcollagen. On the contrary, this denatured collagen is likely to bedecomposed relatively rapidly by collagenase, and cannot maintain itselfsufficiently until new tissues are built up by penetration offibroblasts. Therefore, it is necessary that the medical material of thepresent invention contains not only the denatured collagen but also thecarrier substrate having high resistance against enzymatic decompositionby collagenase or the like, in order to maintain a necessary mechanicalstrength for a certain period in the environments of the living body.This carrier substance is explained below.

The carrier substance used for this purpose is required to be capable ofresisting against the action of decomposing enzyme until the invasion ofcells is sufficiently promoted, and be also biocompatible. Examples ofsuch substance may include polyester, polyurethane, vinyl chloride andother synthetic resins. However, more preferable carrier substances arebiological substances such as collagen, fibroin, polylactic acid,mucopolysaccharides, and arginic acid, which are finally absorbed in theliving body. What is particularly preferable is collagen, and morespecifically preferred is Aterocollagen lacking of the teropeptide whichhas antigenicity.

The collagen used as the carrier substance is, naturally, undenaturedcollagen which has not been subjected to the denaturing treatmentdescribed above. More preferably, it should be a collagen in whichcrosslinking structure is introduced. The method of introducingcrosslinking structure into collagen may comply with the conventionalprocess, for example, the collagen may be heated or treated withcrosslinking agent. In the case of heat treatment, the collagen isheated at 50° to 180° C. for 1 to 24 hours, preferably at 10° to 120° C.for 2 to 8 hours in a vacuum of less than 0.05 Torr, thereby performingthermal dehydration. When treating with crosslinking agent, thecrosslinking agent to be used is not particularly limited. For example,aldehyde crosslinking agents such as glutaraldehyde, isocyanatecrosslinking agents such as hexamethylene diisocyanate, and carbodiimidecrosslinking agents such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride may be used.

The fibrous collagen may be also used preferably as such carriersubstance. The fibrous collagen is prepared by neutralizing the collagencomposed of triple chain helix, by using phosphate buffer at 37° C.Thus, the water-soluble and dispersant triple chain helix structure isreconstructed into fibrous structure having periodicity, and thecollagen is made insoluble.

The crosslinked fibrous collagen obtained by introducing a crosslinkingstructure into the fibrous collagen is particularly preferable carriersubstance. The dispersant triple chain collagen is not particularlyenhanced in the properties such as mechanical strength by introducingcrosslinking structure, but when crosslinking structure is introducedinto fibrillated collagen, the properties such as mechanical strengthare remarkably improved owing to the synergic effects of the fibrousstructure and the crosslinking structure. The crosslinking structure isintroduced after forming the fibrous structure. When the degree ofcrosslinking introduced into the fibrous collagen is too low, sufficientphysical strength will not be obtained. On the contrary, when it is toohigh, the structure and preferable properties of the collagen aresacrificed. Therefore, it is necessary to set appropriately thecrosslinking-degree depending on the conditions. When using acrosslinking agent, an appropriate degree will be obtained at theconcentration of crosslinking agent of, generally, 0.01 to 5% (w/v), orpreferably 1 to 3% (w/v).

With the medical material of the present invention has an excellentcell-penetrating property due to the denatured collagen mentioned above,a sufficient strength may be maintained until corium-like connectivetissues are formed by sufficient cell-penetration because of thecombined use of carrier substance. In other words, the carrier substancein the denatured collagen forms a skeletal structure resisting againstthe decomposition action of enzyme. In addition, when biologicalsubstance such as collagen are used as the carrier substance, thecarrier substance will be finally decomposed by the enzyme action.Therefore, it is completely assimilated in the body. Meanwhile, theratio of denatured collagen to the carrier substance may be about 5 to80% (w/v), or preferably 10 to 50% (w/v).

The medical material of the present invention may be embodied in variousforms depending on the applications, such as film and sponge. In theembodied form, the carrier substance may be merely dispersed in thedenatured collagen. Or, for example, the denatured collagen may beimpregnated in the spongy matrix of the carrier substance.

The method for manufacturing the medical material of the presentinvention is exemplified below. Although collagen is used as carriersubstance in the following method, the same method may be also appliedusing other material than collagen as the carrier substance.

In a first method, first of all, a collagen aqueous solution isprepared, and it is divided into two halves. The first half portion islet stand as it is, and the second half is heated to denature thecollagen. Thus obtained denatured collagen solution and undenaturedcollagen solution are mixed, and are made into a desired form. Forexample, the mixture may be formed into a film by solvent castingmethod, or into a porous sponge by freeze-drying method. Afterwards, asrequired, the film or sponge is subjected to thermal crosslinking orchemical crosslinking.

In a second method, using only the undenatured collagen solutionobtained in the above method, a porous film or porous sponge is formedin the same manner as described above. The film or spongy matrix thusobtained is then subjected to thermal crosslinking or chemicalcrosslinking. Thereafter, the matrix is immersed in denatured collagensolution, and dried in air or in vacuum or freeze-dried.

The ratio of denatured collagen to carrier substance is about 5 to 80%(w/v), or preferably 10 to 50% (w/v).

ARTIFICIAL SKIN

The artificial skin of the invention is a multi-layer complex film usingthe aforementioned cell-penetrable medical material, and typically it isa complex film of three-layer structure as shown in FIG. 1. In FIG. 1,numeral 1 denotes a support layer. A wound contact layer 2 is laminatedon the lower side of the support layer 1, and a moisture penetrationregulating layer 3 is laminated on the upper side of the supportlayer 1. The cell-penetrable medical material is used in the woundcontact layer 2. These layers are individually described below.

(1) Wound contact layer 2

As the wound contact layer 2, the foregoing cell-penetrable medicalmaterial using fibrous collagen as the carrier substance is particularlyemployed. That is, the wound contact layer 2 is composed of denaturedcollagen with the helix content of 0 to 80% and fibrous collagen.Composition and the like of the wound contact layer comprising thesecomponents are omitted herein, because they are already described above.Of course, the wound contact layer, like the medical material mentionedabove, may be formed in desired forms such as film and sponge. Thecarrier substance may be merely dispersed in the denatured collagen. Or,for example, the denatured collagen may be impregnated in the spongymatrix of the carrier substance.

The functions of the wound contact layer 2 are to directly cover thewound surface to protect softly, suppress the pain, give moderatemoisture, and prevent bacterial infection. It is also intended topromote new tissue formation by cell proliferation, and to encouragehealing. The wound contact layer 2 made of such cell-penetrable medicalmaterial possesses all these functions, and it is particularly excellentin promotion of new tissue formation. More, specifically, when appliedon the wound surface, macrophages, neutrophils and other inflammatorycells infiltrate, and fibroblasts invade in early phase. As a result,colium-like connective tissues are built up, and healing of the wound isencouraged. Concerning the invasion of cells, further explanation isomitted herein because it is already described in detail on the medicalmaterial. Finally, the wound contact layer 2 completely decomposed byenzymes and absorbed in the body. Therefore, unlike the conventionalartificial skin, it does not accompany a striking pain upon removingafter healing.

(2) Support layer 1

The support layer 1 is to reinforce the mechanical strength of the woundcontact layer 2, and allow the cells to invade into the wound contactlayer 2 smoothly.

As mentioned above, the wound contact layer possesses a specifiedresistance to collagenase by the combined use of fibrous collagen.However, the mechanical strength for covering material is stillinsufficient, and since it is finally assimilated in the body, itrequires any support body for protecting it from external stimulation.Therefore, the support layer 1 is required to possess a specificmechanical strength. At the same time, the support layer 1 should notimpede invasion of cells into the wound contact layer 2. As the materialfor satisfying these conditions, fibroin is used in the first artificialskin. In the second artificial skin, fibrous collagen with crosslinkingstructure is employed.

Fibroin is a biological material, and is a protein which is a neinconstituent of silk. As known from the fact that silk thread is used assurgical suture, fibroin is a protein excellent in stability in livingbodies. Aqueous solution of fibroin can be prepared by dissolvingfibroin in a concentrated neutral solution of salts such as lithiumbromide or calcium chloride and subjecting the solution to dialysis orthe like. This aqueous solution of fibroin is freezed at -18° C. to 0°C. and defrosted to form β-type crystalline structure, thereby givingwater-insoluble porous unwoven cloth [Jun Umagoshi, Kobunshi Kagaku(Polymer Chemistry), 30, 582 (1973)]. This porous unwoven cloth offibroin shows more excellent stability in living bodies than ordinarycollagen having crosslinking structure. So, it is appropriately used asa supporting layer of artificial skin used for coating the surface ofwould over a long period of time.

The fibrous collagen with crosslinking structure is as mentioned above.That is, when crosslinking structure is introduced to fibrous collagen,the mechanical strength and other properties are markedly enhanced bythe synergistic action of fibrous structure and crosslinking structure.Therefore, it is favorably used as the support layer of artificial skin.

(3) Moisture Permeation Regulating Layer 3

Moisture permeation regulating layer 3 functions to control watercontent on the surface of wound during artificial skin is applied on thesurface of wound. Pooling exudate on the surface of wound can be avoidedwhile keeping the surface of wound wet and moist, by ensuringappropriate steam permeation with this moisture permeation regulatinglayer 3. At the same time, transudation of the protein ingredients inthe exudate outside can be prevented, thereby making favorablecircumstances for repairing tissues. It is heretofore well known thatsuch moisture permeation regulating layer is used in wound coatingmaterial, and these known materials can be also used for artificial skinof the present invention. Thus, a film of non-toxic material havingabout 0.1 to 1 mg/cm² /hour of water flux can be used as such moisturepermeation regulating layer 3. Its appropriate thickness is about 5 to200 μm. Non-toxic materials include silicone resin, polyacrylate ester,polymethacrylate ester, polyurethane and the like. In particular,silicone resin is preferred.

Said artificial skin having three layers can exhibit very excellenttherapeutic effect as artificial skin, because wound-contacting layer 2has favorable cell-penetration and appropriate resistance againstenzymic decomposition, satisfactory mechanical strength is given bysupporting layer 1 and appropriate moisture content is kept on thesurface of wound by moisture permeation regulating layer 3. Since animprovement of cell-penetration and resistance to enzymes, both of whichwere heretofore incompatible, have been attained in wound-contactinglayer 2, there can be obtained by far more excellent therapyaccelerating effect than known wound coating materials.

In preferred embodiments of said first and second artificial skin,antibacterial agent is incorporated in at least one of suchwound-contacting layer 2, supporting layer 1 and moisture permeationregulating layer 3. Appropriate antibacterial agents include silversulfadiazine, gentamycin and silver nitrate. However, various otherantibacterial agents can be used without limiting to them. Incorporationof such anti-bacterial agents is a means for preventing effectivelyantibacterial infection.

Bacterial infection is apt to occur in broad ambustion or seriousambustion (for example, degree III ambustion). Cream base containingantibacterial agent has heretofore been used for preventing suchbacterial infection. However, when cream base is used together withknown wound coating material such as gauze or bandage, about 57% of theapplied cream soaks into gauze or the like together with the exudate andabout 21% of the cream only gets to the surface of wound. Further, thecream has to be applied everyday to need troublesome labour. On thecontrary, if the artificial skin of the present invention containsantibacterial agent, it can release gradually the antibacterial agent ina certain period. Therefore, the antibacterial agent can actcontinuously without exposing the surface of wound to ambient air andwithout troublesome labour in applying the antibacterial agent to thewound everyday, whereby bacterial infection can be prevented effectivelyin combination with bacterial penetration-preventive effect of themoisture permeation generating layer.

The following experiment was performed for examining the aspect ofreleasing antibacterial agent contained in moisture permeationregulating layer 3. At first, three silicone films (about 0.15 g) of 20μm thick in size (5 cm×5 cm) were prepared, which respectively contain10 mg, 20 mg and 30 mg of silver sulfadiazine (AgSD). These respectivesamples were dipped in 100 ml of distilled water, and the amount of AgSDeluted was measured in the lapse of time, respectively. The result isshown in FIG. 2. In this Figure, the abscissa shows the lapse of time(day) and the ordinate shows the cumulative amount of AgSD eluted. It isobserved from the result that AgSD contained in the silicone film isgradually released to attain the gradual releasability enough to get theeffect described above.

When a main object is to prevent bacterial invasion from outside and itis necessary to add antibacterial agent into any one of these layers, itis preferred to select moisture permeation regulating layer 3. On theother hand, when the surface of wound is already infected by bacteriaand a lot of antibacterial agent is needed to be applied on the wound,it is desirable to incorporate antibacterial agent into eitherwound-contacting layer 2 or supporting layer 1. Of course, one canincorporate antibacterial agent into any two or three members ofwound-contacting layer 2, supporting layer 1 and moisture permeationregulating layer 3.

MANUFACTURING METHOD OF ARTIFICIAL SKIN

The manufacturing method of artificial skin of the invention is aprocess for efficiently preparing either first or second artificialskin, having a wound contact layer in which the carrier substance ismerely dispersed in the denatured collagen. This method may be easilyrealized by the following steps a) to f).

a) In the first place, a collagen aqueous solution is prepared, and isdivided into two halves. One half portion is let stand as it is, whilethe other half portion is heated to denature the collagen. Thus obtaineddenatured collagen solution and undenatured collagen solution aremised.The obtained mixed solution is used for forming the wound contact layer2.

b) Separately, fibroin film or fibrous collagen film with crosslinkingstructure, which is used as the support layer 1, is formed.

The fibrous collagen film with crosslinking structure is formed into aporous spongy film by freeze-drying the aqueous solution of crosslinkedfibrous collagen prepared by the previously mentioned method.

The fibroin film is also manufactured into porous film by the methodalready described.

c) In the next step, the mixed solution is received in a certaincontainer, and the fibroin film or the fibrous collagen film withcrosslinking structure is put on the liquid surface, and isfreeze-dried. As a result, a laminate of the support layer 1 and woundcontact layer 2 is obtained. In this case, the support layer 1 is afibroin film or a fibrous collagen film with crosslinking structure, andthe wound contact layer 2 is a porous layer containing denaturedcollagen and fibrous collagen.

d) On a substrate having a peeling surface such as Teflon substrate, asolution of a substance such as silicone for affording moisturepermeable film is developed, and a viscous thin film is formed.

e) On the viscous thin film, the laminate of support layer 1 and woundcontact layer 2 is placed in such manner that the support layer 1contacts with the viscous thin film.

f) After the viscous thin film is dried until hardened, in a vacuum ofless than 0.05 Torr, it is further heated for 1 to 24 hours at 50° to180° C. As a result, the viscous thin film is dried, and the moisturepermeation regulating layer 3 is formed. At the same time, theregulating layer 3 is integrally bonded on the support layer 1. Thus isobtained the artificial skin in three-layered structure as shown in FIG.1.

In this way, according to the method of the invention, without using anyadhesives, an artificial skin in laminate structure as shown in FIG. 1may be easily manufactured.

Meanwhile, a preferred artificial skin containing antibacterial agentmay be manufactured by incorporating an antibacterial agent at leastinto one of the mixed solution for forming the wound contact layer 2,the fibroin film or the fibrous collagen film with cross-linkingstructure to be used as support layer 1, or the solution containing thesubstance for offering the moisture permeation regulating layer 3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sectional view showing an example of a multilayer structurein an artificial skin of the present invention; and

FIG. 2 is a graph showing the results of investigation of the releaserate of the antibacterial agent contained in the moisture penetrationregulating layer in the artificial skin in FIG. 1.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is now described in further details by way of examples.

<A> EXAMPLES OF CELL-PENETRABLE MEDICAL MATERIAL (EXAMPLES 1 to 4)Example 1: Preparation of Aterocollagen/denatured Aterocollagen Matrix

1.0 g of aterocollagen (AC) was dissolved in diluted hydrochloric acidat pH3.0.

This solution was kept in a 60° C. thermostatic oven for 30 minutes, andwas left stand at room temperature for 2 hours to obtain a solution ofdenatured aterocollagen (HAC). The helix content of thus obtained HACwas about 40%. While stirring 0.3 w/v % AC (pH3.0) solution, 0.3 w/v %HAC solution was added to the AC solution and mixed. This solution waspoured into a stainless steel vat, and was directly frozen quickly to-30° C., and after sufficient freezing, it was freeze-dried in a vacuumof less than -40° C./0.1 Torr. The product was thermally dehydrated andcrosslinked by heat treating for 24 hours at 110° C. in a vacuum of lessthan -40° C./0.1 Torr.

Reference 1: Preparation of AC Matrix

1.0 g of AC was dissolved in diluted hydrochloric acid at pH3.0 up tothe concentration of 0.3 w/v %. This solution was freeze-dried in thesame manner as described above, and was further heated, dehydrated andcrosslinked.

Test 1: In Vitro Cell Invasion Test of AC/HAC Matrix

Cell invasion into matrices obtained in Example 1 and Reference 1 wasevaluated by conducting in vitro culture test using rat skinfibroblasts.

A collagen sponge of 3.5 cm in diameter was put on a 60 mm sterilizedPetri dish (manufactured by Termo), and fibroblasts were dropped on thesponge by 1 ml at a concentration of 1×10⁶ cells/ml, and cultivated for24 hours at 37° C. Furthermore, 3 ml of DME culture medium containing10% FBS was added and cultivated for 6 days at 37° C.

After fixing in 10% neutral buffer malin solution, the specimen wasstained and observed by optical microscope. The results of evaluationare shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        In vitro cell invasion test                                                   into collagen matrix                                                                           Cell     Sponge from                                         Specimen         invasion retaining ability                                   ______________________________________                                        AC               -        +++                                                 AC - 20 wt. % HAC                                                                              ±     +++                                                 AC - 33 wt. % HAC                                                                              +++      +++                                                 AC - 50 wt. % HAC                                                                              +++      +++                                                 AC - 67 wt. % HAC                                                                              +++      +++                                                 AC - 80 wt. % HAC                                                                              +++      +                                                   ______________________________________                                        Note:                                                                                  Cell invasion                                                                              Sponge form retaining ability                           ______________________________________                                        -        None         Eliminated (dissolved)                                  ±     Slight invasion                                                                            Almost dissolved                                        +        Small invasion                                                                             Extreme deformation although                                                  specimen remained                                       ++       Medium invasion                                                                            Slight contraction, dissolving                          +++      Marked invasion                                                                            Unchanged                                               ______________________________________                                    

It is known from Table 1 that the matrix mixed with HAC was notablyimproved in the cell invasion as compared with the matrix of AC alone.However, from the viewpoint of retaining the sponge shape, it seemsdesired to contain the HAC by less than 80 wt. %.

Reference 2: Preparation of Fibrous AC

1.0 g of AC was dissolved in diluted hydrochloric acid at pH3.0 toobtain 0.3 w/v % solution. While stirring this solution in 4° C.thermostatic oven, phosphate buffer was added into the solution toprepare collagen solution with the final concentration of 0.1% (w/v)aterocollagen, 30 mM disodium phosphate, 100 mM NaCl. The collagensolution was immersed in 37° C. thermostatic bath for a day, therebyobtaining fibrous collagen (FC) solution. This solution was centrifuged(5000 rpm, 10 minutes), and concentrated, and 0.3% (w/v) fibrousaterocollagen (FC) solution was prepared. This solution was quicklyfrozen at -30° C., and then freeze-dried to prepare sponge. This spongewas heated in a vacuum at 110° C. for 2 hours to be dehydrated andcrosslinked.

Example 2: Preparation of Fibrous Aterocollagen/denatured AterocollagenMatrix

Mixing the 0.3% (w/v) FC solution and the 1% (w/v) HAC solution preparedin Example 1 at 37° C., the mixture was stirred for an hour. Thissolution was quickly frozen at -30° C., and was freeze-dried to preparesponge. Afterwards, the sponge was treated in a vacuum at 110° C. for 2hours to be heated, dehydrated and crosslinked.

Test 2: In Vitro Cell Invasion Test of Fibrous Aterocollagen/denaturedAterocollagen Matrix

Cell invasion into the matrices obtained in Example 2 and Reference 2was evaluated, by conducting in vitro culture experiment using ratfibroblasts in the same operation as in Test 1. The results ofevaluation are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        In vitro cell invasion test                                                   into collagen matrix                                                                          Cell     Sponge from                                          Specimen        invasion retaining ability                                    ______________________________________                                        FC              +        +++                                                  FC - 10 wt. % HAC                                                                             ++       +++                                                  FC - 20 wt. % HAC                                                                             +++      +++                                                  FC - 50 wt. % HAC                                                                             +++      +++                                                  ______________________________________                                    

As shown in Table 2, the matrices comprising FC were all excellent inthe sponge form retention, and superior in stability. As for invasion ofcells, although a slight deviational cell invasion was noted in FCalone, many cells invaded uniformly and dispersantly in the HAC addedseries. Further, the sponge shape of HAC added series was close to thein vivo tissues, in spite of the in vitro culture experimental system.

Test 3: In Vivo Subcutaneous Embedding Test of FibrousAterocollagen/denatured Aterocollagen Matrix

The matrices prepared in Example 2 and Reference 2 were embedded in therat skin, and tissue images were researched pathologically.

For subcutaneous transplantation (embedding) female Wistar KY ratsweighing about 200 g were used. Before embedding, the rats wereanesthetized with Nembutal diluted five times, and the dorsal hairmoistened with surgical Isodine (Meiji Seika) was carefully clippedcompletely by a clipping razor. The clipped dorsal surface wasdisinfected with Isodine and ethanol. From each cut, the incision iswidened so as to form a free gap in the areola tissues beneath thedermal muscle of rat (the adjacent cuts should not communicate with eachother). The specimen was inserted into the gap, and was laid downflatly. The wound is sutured by nylon thread with corner needle. Thewound was stitched with three sutures. The same specimen was embeddedsimilarly in other rat.

On the 3rd day and the 28th day after embedding, the animals were killedby using ether or Nembutal diluted twice. Dermal tissues on the dorsalmuscle of rat was cut out in a size of 8 cm by 12 cm or larger, keepingthe embedded specimen in tissues. The tissue was put in 10% neutralbuffer formalin solution, and let stand overnight to be fixed, and waspresented for histopathological examination.

Histopathological search was started by cutting out the specimen fromthe tissues. In order that the specimen be securely contained, thetissues were cut in a strip of about 0.5 cm by 2.5 cm. It was penetratedthrough ethanol and then xylene, and was finally replaced by paraffin.After replacing, the tissues containing the specimen were put in a hotmolten liquid of solid paraffin, and cooled quickly to finish paraffinembedding. The paraffin embedded tissues were sliced by means of rotarymicrotome of Yamato, and 4 μm thick paraffin segments were obtained.After removing paraffin, the specimen was stained histopathologically inan arbitrary staining method to complete the preparation. As thehistopatological staining, hematoxylineosin (HE) staining, azanestaining, resorsin-fuchsin staining, and other methods may be employed.The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Histopatological changes in subcutaneous embedding test                                 Tissue changes                                                                Day 3          Day 28                                                           Neutrophil Fibroblast                                                                              Granulation                                  Specimen    infiltration                                                                             invasion  tissue atrophy                               ______________________________________                                        FC          +++        ++        +++                                          FC - 10 wt. % HAC                                                                         +          +++       +                                            FC - 20 wt. % HAC                                                                         ++         +++       +                                            FC - 50 wt. % HAC                                                                         +++        ++        +                                            ______________________________________                                    

With FC alone, on the 3rd day, the neutrophil infiltration was intense,and fibroblast invasion was moderate, and on the 28th day, the formedgranulation tissues were atrophied. By contrast, when HAC was containedby 10 or 20 wt. %, on the 3rd day, the neutrophil infiltration was weak,while the fibroblast invasion was more smooth. On the 28th day, theatrophy of granulation tissues was notably lessened.

Example 3: Preparation of Crosslinked Collagen Coated with DenaturedCollagen

The freeze-dried sponge of fibrous aterocollagen (FC) obtained inReference 2 was immersed overnight in 0.01% and 1%hexamethylene-di-isocyanate (HDI)/ethanol solution, and chemicalcrosslinking was introduced. To each sponge, the aqueous solution ofdenatured collagen (HAC) obtained in Example 1 was added by 30 ml. Aftersufficient immersion, it was freeze-dried again to obtain sponge, whichwas heated for 2 hours in a vacuum at 110° C., and was further heatedfor 24 hours to be dehydrated and crosslinked. Thus, the collagen matrixcoated with denatured collagen (HAC) was obtained. The final compositionwas adjusted so that the content of HAC be 10 wt. %.

Reference 3: Preparation of Crosslinked Collagen

The freeze-dried sponge of fibrous aterocollagen (FC) alone (crosslinkedin the same procedure as in Example 3) was prepared as Reference 3, inthe same manner as Example 3 except that the step of addition of aqueoussolution of denatured collagen (HAC) was omitted.

Test 4: In Vivo Subcutaneous Embedding Test of Denatured Collagen CoatedCrosslinked Collagen Matrix

The matrices prepared in Example 3 and reference 3 were embedded beneaththe rat skin in the same procedure as in Test 3, and histopathologicalexaminations were conducted. However, the specimens were taken out on7th day and 14th day. The results are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Histopatological changes in subcutaneous embedding test                                       Tissue changes on 7th day                                                     and 14th day                                                                        Neutrophil                                                                              Fibro- Self-                                          Crosslinking  infiltra- blast  tissue                                 Specimen                                                                              condition     tion      invasion                                                                             making                                 ______________________________________                                        FC      0.01% HDI cross-                                                                            ++        -      -                                              linking + thermal                                                             crosslinking 2 hrs                                                    HAC     0.01% HDI cross-                                                                            +         +++    +++                                    coated FC                                                                             linking + thermal                                                             crosslinking 2 hrs                                                    FC      1% HDI cross- ±      -      -                                              linking + thermal                                                             crosslinking 2 hrs                                                    HAC     1% HDI cross- +         +      +                                      coated FC                                                                             linking + thermal                                                             crosslinking 2 hrs                                                    FC      1% HDI cross- -         -      -                                              linking + thermal                                                             crosslinking 24 hrs                                                   HAC     1% HDI cross- ++        ++     ++                                     coated FC                                                                             linking + thermal                                                             crosslinking 24 hrs                                                   ______________________________________                                    

In each comparative example of FC, although there was partly invasion ofneutrophils, the infiltration of the cell components themselves was verypoor, including the inflammatory and reticuloendothelial cells. Bycontrast, in each HAC coated FC, the infiltration of cell components wasvery smooth, and partly self-tissue making was noted while foreignmatter reaction was slightly strong in some cases. Particularly, in thespecimen of HAC coated FC (0.01% HDI crosslinking+thermal crosslinking 2hours), a structure extremely similar to the corium in which even theneutrophil infiltration has been already reduced was rebuilt. Thus, itmay be regarded as a nearly ideal matrix in view of the objects of thepresent invention.

Example 4: Preparation of Collagen Sponge Containing Silicone Film

On a teflon substrate, a hexane solution of 50% Silastic siliconeadhesive type A (Dow Corning) was applied by using a precise applicatorto form a film. Immediately after application, the sponge manufacturedaccording to Example 3 was put on, and left stand at room temperaturefor about 10 minutes, and was hardened in an oven for at least an hourat 60° C.

Test 5: Transplantation Test in Skin Defect Wound

The sponge prepared in Example 4 was transplanted in a skin defect woundof rat. More specifically, in the dorsal skin of rat, a total skindefect wound (2 cm by 2 cm) with an exposed subcutaneous muscularmembrane was prepared, and the specimen having a silicone film adheredon the superficial layer was ligated and sutured on the wound surface.The animals were killed in 4 weeks after transplantation, and thetransplanted material and wound bed were cut out, and presented forpathological examinations. In 4 weeks, contraction of wound was notclearly observed, and a favorable granulation tissue was formed andregeneration of the cuticle was recognized.

<B> EXAMPLES OF PROSTHETIC SKIN AND ITS MANUFACTURING METHOD Example5 1) Preparation of wound contact layer

Preparation of fibrous collagen/denatured collagen matrix

1.0 g of AC was dissolved in diluted hydrochloric acid at pH3.0 to 0.3w/v %. While stirring this solution in a 4° C. thermostatio oven,phosphate buffer was added to prepare collagen solution with the finalconcentration of 0.1 w/v % aterocollagen, 30 mM phosphate-2-sodium, 100mM NaCl. Immersing overnight in a 37° C. thermostatic oven, fibrouscollagen (FC) solution was obtained. This solution was centrifuged (5000rpm, 10 minutes) and concentrated, and 0.3 w/v % fibrous aterocollagen(FC) solution was prepared. On the other than, 1.0% aterocollagen (pH3.0hydrochloric acid) solution was treated for 30 minutes in a 60° C.therostatic oven, and was left stand at room temperature for 2 hours,and a solution of denatured aterocollagen (HAC) was obtained. Thusprepared solutions were mixed at 37° C., and stirred for an hour. Thismixed solution was quickly frozen at -30° C., and freeze-dried toprepare sponge.

2) Preparation of fibroin matrix

Refined silk was dissolved in 8M lithium bromide aqueous solution, andthe solution was put into a cellophane tube to be presented for dialysisin water. After confirming complete removal of lithium bromide, theobtained fibroin aqueous solution was poured into a polystyrenecontainer, and was frozen for 24 hours at -10° C. Thereafter, thawing atroom temperature, the nonwoven state was confirmed, and it wasfreeze-dried.

3) Preparation of wound covering

The mixed solution of collagen/denatured collagen obtained in 1) waspoured into a stainless steel vat, and the sponge of the matrix offibroin prepared in 2) was put on slowly, and in this state the materialwas quickly frozen at -30° C. and after sufficiently freezing it wasfreeze-dried, and a sponge of two-layer structure was obtained. Next, ona teflon substrate, a hexane solution of 50% Silastic silicone adhesivetype A (Dow Corning) was applied by using a precise applicator, and afilm was formed. Just after application, the sponge was put on the filmso that the fibroin matrix contact to the silicone side, and afterletting stand at room temperature for about 10 minutes, it was hardenedin an oven for at least one hour at 60° C. Furthermore, keeping in avacuum for an hour, the temperature was raised to 110° C., and thevacuum was maintained for 2 hours, then the temperature was lowered toroom temperature, and the specimen was taken out, and the wound coveringwas obtained.

Example 6

In step 3) of Example 5, 25 mg of silver sulfadiazine was added to 50 ccof the mixed solution of collagen and denatured collagen, and themixture was stirred sufficiently, and poured into a stainless steel vat.Slowly putting a matrix of fibroin, the material was quickly frozensufficiently at -30° C., and freeze-dried, and a two-layer spongecontaining antibacterial agent was obtained. By similarly laminating asilicone film on this sponge, a wound covering with antibacterial agentwas obtained.

Example 7 1) Preparation of fibrous atelocollagen

1.0 g of atelocollagen was dissolved in diluted hydrochloric acid atpH3.0 to 0.3 w/v %. This solution was put in a 4° C. thermostatic ovenand stirred, and phosphate buffer was added to prepare collagen solutionwith the final concentration of 0.1 w/v % atelocollagen, 30 mMphosphate-2-sodium, 100 mM sodium chloride. Immersing in 37° C.thermostatic bath for a day, fibrous atelocollagen (FC) solution wasprepared. This solution was quickly frozen at -30° C., and freeze-driedto obtain sponge.

2) Preparation of denatured atelooollagen solution

1.0 g of atelocollagen was dissolved in diluted hydrochloric acid atpH3.0. This solution was treated for 30 minutes in 60° C. thermostaticoven, and was left stand at room temperature for 2 hours to obtaindenatured atelocollagen (HAC). The helix content of thus obtaineddenatured atelocollagen was about 40%.

3) Preparation of fibrous atelocollagen/denatured atelocollagen matrix

The above prepared 0.3 w/v % fibrous atelocollagen (FC) and 1 w/v %denatured atelocollagen (HAC) were mixed at 37° C., and stirred for anhour. This solution was quickly frozen at -30° C., and freeze-dried toprepare sponge.

4) Thermal dehydration crosslinking of fibrous atelocollagen

The product of 1) was evacuated for an hour in a vacuum of less than0.05 Torr, and the temperature was lowered to 110° C., and the vacuumwas maintained for 2 hours. Then, the temperature was lowered to roomtemperature, and the specimen was taken out.

5) Isocyanate crosslinking of fibrous atelocollagen matrix

The product of 1) was crosslinked by immersing in ethanol solution of0.01% hexamethylene diisocyanate (HDI) for 24 hours at room temperature.It was washed in water several times to get rid of the uncrosslinkedisocyanate, and freeze-dried.

6) Thermal dehydration crosslinking of fibrous atelocollagen/denaturedatelocollagen matrix

The product of 3) was evacuated for an hour in a vacuum of less than0.05 Torr, and the temperature was lowered to 110° C. and the vacuum wasmaintained for 2 hours or 24 hours. Then, the temperature was lowered toroom temperature, and the specimen was taken out.

7) In vivo subcutaneous embedding test of collagen matrices (Test 6)

The matrices obtained herein were embedded beneath the rat skin, and thetissue images were studied pathologically. For subcutaneoustransplantation, female Wistar KY rats weighing about 200 g were used.Before embedding, the rats were anesthetized with Nembutal diluted fivetimes, and the dorsal hair moistened with surgical Isodine (Meiji Seika)was carefully clipped completely by a clipping razor The clipped dorsalsurface was disinfected with Isodine and ethanol. From each cut, theincision is widened so as to form a free gap in the areola tissuesbeneath the dermal muscle of rat. The specimen was inserted into thegap, and was laid down flatly. The wound is sutured by nylon thread withcorner needle. The wound was stitched with three sutures. The samespecimen was embedded similarly in other rat.

On the 3rd day and the 28th day after embedding, the animals were killedby using ether or Nembutal diluted twice. The dermal tissues on thedorsal muscle of rat was cut out in a size of 8 cm by 12 cm or larger,keeping the embedded specimen in tissues. The tissue was put in 10%neutral buffer formalin solution, and left stand overight to be fixed,and was presented for histopathological examination.

Histopathological search was started by cutting out the specimen fromthe tissues. In order that the specimen be securely contained, thetissues were cut in a strip of about 0.5 cm by 2.5 cm. It was penetratedthrough ethanol and then xylene, and was finally replaced by paraffin.After replacing, the tissues containing the specimen were put in a hotmolten liquid of solid paraffin, and cooled quickly to finish paraffinembedding. The paraffin embedded tissues were sliced by means of rotarymicrotome of Yamato, and 4 μm thick paraffin segments were obtained.After removing paraffin, the specimen was stained histopathologically inan arbitrary staining method to complete the preparation. As thehistopatological staining, hematoxylin-eosin (HE) staining, azanestaining, resorsin-fuchsin staining, and other methods may be employed.The results are shown in Table 5.

                                      TABLE 5                                     __________________________________________________________________________    Histopatological changes in subcutaneous embedding test                                                    Tissue changes                                                                Day 3       Day 28                                                            Neutrophil                                                                          Fibroblast                                                                          Granulation                          Specimen  Crosslinking method                                                                              infiltration                                                                        invasion                                                                            tissue atrophy                       __________________________________________________________________________    FC        0.01% HDI (Room temperature 24 H)                                                                -     -     -                                    FC        Thermal dehydrating crosslinking                                                                 +++   ++    +++                                            (110° C., 2 H)                                               FC-10 wt. % HAC                                                                         Thermal dehydrating crosslinking                                                                 +     +++   +                                              (110° C., 2 H)                                               FC-20 wt. % HAC                                                                         Thermal dehydrating crosslinking                                                                 ++    +++   +                                              (110° C., 2 H)                                               FC-50 wt. % HAC                                                                         Thermal dehydrating crosslinking                                                                 +++   ++    +                                              (110° C., 2 H)                                               FC-10 wt. % HAC                                                                         Thermal dehydrating crosslinking                                                                 +++   +     +++                                            (110° C., 24 H)                                              __________________________________________________________________________     -: negative, +: slight, ++: moderate, +++: severe HAC treated for 30          minutes at 60° C.                                                 

With FC alone, on the 3rd day, the neutrophil infiltration was intense,and fibroblast invasion was moderate, and on the 28th day, the formedgranulation tissues were atrophied. By contrast, when HAC was containedby 10 or 20 wt. %, on the 3rd day, the neutrophil infiltration was weak,while the fibroblast invasion was more smooth. On the 28th day, theatrophy of granulation tissues was notably lessened.

8) Preparation of prosthetic skin

In 3), the concentration of fibrous atelocollagen was adjusted to 1.0w/v %, and the mixed solution of fibrous atelocollagen/denaturedatelocollagen was poured into a stainless steel vat, and the sponge offibrous atelocollagen matrix crosslinked with 0.0.% HDI prepared in 5)was slowly put on, thereby floating the sponge in the upper layer of thesolution. In this state, the solution was quickly frozen at -30° C., andfrozen sufficiently, and was freeze-dried in a vacuum of less than -40°C./0.1 Torr, thereby obtaining a sponge of two-layer structure composedof a fibrous atelocollagen matrix and a matrix of fibrousatelocollagen/denatured atelocollagen. Next, on a teflon substrate,hexane solution of 50% Silastic silicone adhesive type A (Dow Corning)was applied by using a precise applicator, and a film was formed. Justafter application, the sponge was put on so that the fibrousatelocollagen contact to the silicone film, and after letting stand atroom temperature for about 10 minutes, it was hardened in an oven for atleast an hour at 60° C. In a vacuum of less than 0.05 Torr, it wasevacuated for an hour, and the temperature was raised to 110° C., andthe vacuum was maintained for 2 hours. Then, the temperature was loweredto room temperature, and the specimen was taken out to obtain prostheticskin.

9) Transplantation test of artificial skin on rat skin defect wound(Test 7)

The matrix obtained above was transplanted on the dorsal skin of rat.The dorsal hair of Wistar KY rats (200 to 400 g) was clipped underNembutal anesthesia. After disinfecting the skin with Isodine, a totalskin defect wound of 20 by 20 mm was prepared by exposing the dermalmuscle on the rat dorsal. Then, after hemostatic process and drying, thespecimen containing physiological saline was adhered. The periphery ofsilicone film was ligated and fixed at 16 positions by suture. Fourpieces of Solfren (Termo) were laid over, and elastic tape such asElasticon was wound around to fix oppressively. The wound site wasobserved 1, 2, 3, 4 weeks later, and the animals were killed for autopsyat the day 4 weeks after transplantation. Results of histopathologicalexaminations are shown in Table 6.

                                      TABLE 6                                     __________________________________________________________________________    Gross and pathological findings in transplantation test                                                            Tissue changes                           Specimen                             4 weeks later                            Upper layer                                                                             Lower layer                                                                              HAC degeneration                                                                        Wound Granulation                                                                           Cuticle                          matrix    materix    condition contraction                                                                         tissue  formation                        __________________________________________________________________________    FC (0.01% HDI                                                                           FC-10 wt. % HAC                                                                           60° C., 30 min                                                                  Weak  Granulation                                                                           Present                          crosslinking)                                                                           (thermal dehydration       resembling                                         crosslinking 2 hrs)        falce corium *1                          FC (0.01% HDI                                                                           FC-10 wt. % HAC                                                                          100° C., 24 H                                                                    Weak  Granulation                                                                           Present                          crosslinking)                                                                           (thermal dehydration       resembling                                         crosslinking 2 hrs)        falce corium                             FC (0.01% HDI                                                                            --         --       Weak  Inflammatory                                                                          None                             crosslinking)                        granulation *2                           FC-10 wt. % HAC                                                                          --         60° C., 30 min                                                                  Intense                                                                             Simple  Present                          (thermal                             granulation *3                           dehydrating                                                                   crosslinking)                                                                 No treatment                                                                             --         --       Very  Simple  Present                                                         intense                                                                             granulation                              __________________________________________________________________________     *1: Composed of curved thick collagen fiber flux, having the gaps             diffusely scattered therein with capillary vessels and fibro blasts.          *2: Compound of residual collagen fibers, giant cells, histocytes and         fibroblasts phagocytizing them, and slight collagen produced by them.         *3: Granulation tissues mainly composed of fibroblasts, and sparse            collagen fibers of simple structure produced by them.                    

EXAMPLE 8 1) Preparation of collagen sponge containing antibacterialagent

Phosphate buffer was added to 0.3% atelocollagen solution at pH3.0, andthe mixture was put in a 37° C. thermostatic oven for 4 hours to preparefibrous atelocollagen. While stirring 50 cc of fibrous atelocollagen, 25mg, 50 mg, 250 mg, and 500 mg of powder of silver sulfadiazine wereadded, and dispersed sufficiently. The mixture was poured into styrolvat (10 cm×10 cm), and freeze-dried. The prepared sponge was crosslinkedfor 2 h ours at 110° C. in a vacuum.

2) Preparation of collagen sponge containing antibacterial agent

Phosphate buffer was added to 0.3% atelocollagen solution at pH3.0, andthe mixture was put in a 37° C. thermostatic oven for 4 hours to preparefibrous atelocollagen. This solution was freeze-dried to form sponge,and further it was crosslinked by immersing for a day in an ethanolsolution of 0.01 hexamethylene isocyanate. The crosslinked collagensponge (10 cm×10 cm) was dipped in 100 ml of ammonia solution of silvernitrate (1×10⁻³ mol/liter).

3) Preparation of collagen sponge containing antibacterial agent

The collagen sponge immersed in ammonia solution of silver nitrateobtained in Example 2 was further immersed in 100 ml of sodiumsulfadiazine solution (1×10⁻³ mol/liter).

4) Evaluation of antibacterial activity (Test 8)

Muller-Hinton agar (Difco) was treated in autoclave and maintained at50° C., and 20 ml thereof was dispensed in Petri dish, and was leftstand at room temperature for an hour to be solidified. The variousbacteria cultivated on a flat disc were suspended in tris buffersolution to prepare bacterial solutions, which were applied on theentire surface of the each culture medium three times by using a swab.Specimens prepared in the previous steps were cut out in a size of 8 mmin diameter, and put on the culture medium coated with bacteria, andcultivated for 18 hours at 37° C. The results are shown in Table 7.

All strains formed nearly equal inhibition circles, not depending on theconcentration of the AgSD, when addition amount is 0.25 mg/cm² or more.

                                      TABLE 7                                     __________________________________________________________________________    Antibacterial activity of collagen sponge containing antibacterial agent              Speciment                                                                     Unit: mm                                                                      AgSD  AgSD   AgSD  AgSD  AgSD        Ag(NH.sub.3).sub.2 --            Species 0 mg/cm.sup.2                                                                       0.25 mg/cm.sup.2                                                                     0.5 mg/cm.sup.2                                                                     2.5 mg/cm.sup.2                                                                     5.0 mg/cm.sup.2                                                                     Ag(NH.sub.3).sub.2                                                                  NaSD                             __________________________________________________________________________    Ps. aeruginosa                                                                        0     11.64  11.64 10.43 11.64 9.54  10.68                                    0     11.52  10.85 10.50 12.32                                        St. epidermidis                                                                       0     12.59  14.04 12.96 13.17 9.67  9.51                                     0     12.93  12.39 12.67 13.22                                        E. coli 0     11.38  12.17 11.74 10.80 9.52  10.32                                    0     12.78  12.96 11.58 11.65                                        C. albicans                                                                           0     17.13  17.23 15.57 17.15 8.34  9.31                                     0     17.81  15.58 15.55 16.03                                        __________________________________________________________________________

5) Preparation of artificial skin containing antibacterial agent

In Example 1, the fibrous atelocollagen solution was adjusted to 1.0 w/v%, and denatured atelocollagen solution was added. Then, 25 mg of silversulfadiazine was added while stirring sufficiently, and the mixture waspoured into a styrol vat. The fibrous atelocollagen crosslinked with0.01% HDI was put on slowly, and freeze-dried. Next, on a teflonsubstrate, hexane solution of 50% Silastic silicone adhesive type A (DowCorning) was applied by using a precise applicator, and a film wasformed. Just after application, the sponge was put on, and was hardenedin an oven for at least an hour at 60° C. Furthermore, evacuating for anhour in a vacuum, and the temperature was further raised to 110° C., andthe vacuum was maintained for 2 hours. Then, the temperature was loweredto room temperature, and the specimen was takeout to obtain a artificialskin containing antibacterial agent.

What is claimed is:
 1. A cell-penetrable medical material, comprisingdenatured collagen having a triple chain helix content of 0 to 80%, anda carrier possessing higher resistance to enzymatic decomposition thanthe denatured collagen.
 2. The medical material of claim 1, wherein thedenatured collagen lacks an antigenic portion at a molecular terminus.3. The medical material of claim 1, wherein the carrier is selected froma group consisting of collagen, fibroin, polyactic acid,mucopolysaccharide, and arginic acid.
 4. The medical material of claim3, wherein the carrier is undenatured collagen.
 5. The medical materialof claim 3, wherein the carrier is collagen lacking an antigenic portionat a molecular terminus.
 6. The medical material of claim 3, wherein thecarrier is collagen with a crosslinked structure.
 7. The medicalmaterial of claim 3, wherein the carrier is fibrous collagen.
 8. Themedical material of claim 7, wherein the fibrous collagen has acrosslinked structure.
 9. The medical material of claim 1, wherein thematerial is a film.
 10. The medical material of claim 1, wherein thecarrier is dispersed in the denatured collagen.
 11. The medical materialof claim 1, wherein the material is a sponge.
 12. The medical materialof claim 11, wherein the carrier forms a spongy matrix, and thedenatured collagen is contained in the spongy matrix.
 13. The medicalmaterial of claim 1, wherein the ratio of denatured collagen to carrieris in the range of 5 to 80% w/v.